The use of electrospray ionisation in mass spectrometry has become commonplace and in particular its use in the study of large biomolecules has become ubiquitous. Although advantageous, as it allows the study of labile species in their complete form, multiple charging can lead to complex mass-to-charge ratio spectra. Since these spectra are observations of ion counts at different mass-to-charge ratios, this multiple charging can result in the parent molecule exhibiting multiple peaks making the parent mass difficult to determine. Consequently a number of methods have been developed to garner information about the mass of the parent molecule from these complex spectra. To extract this information, knowledge regarding the charge states of the observed parent molecule ions is required. Using high resolution spectra (those with isotopic resolution) the assignment of these charge states can be easily inferred from the separations between adjacent isotopic peaks. In low resolution mass-to-charge ratio spectra (those without isotopic resolution) the extraction of the parent molecule mass is not as straightforward.
The use of low resolution mass spectrometry for the study of large molecules such as biomolecules does present certain advantages. These advantages stem from the ability to produce lower resolution mass spectrometry instruments at reduced cost and the miniaturisation of such instruments is also easier. These benefits make the use of low resolution instruments for on-line and at-line analysis very appealing. This use of mass spectrometry presents additional challenges as sampling is often from a range of environments with little or no sample preparation. A result of this is that mass-to-charge ratio spectra can appear noisy and can contain extraneous peaks caused by solvent clusters and ion adducts originating from the sample media. The extraction of parent molecule masses in these scenarios can therefore be very challenging. In particular, techniques relying on peak picking can easily fail when strong extraneous peaks and poor signal to noise are characteristics of the mass-to-charge ratio spectra. In addition, peak picking by its nature results in the reduction of a peak to a single mass-to-charge ratio. Peak picking techniques can therefore lead to the misrepresentation of peaks with shoulders or any form of asymmetry. Other deconvolution methods can be strongly influenced by background noise, leading to the generation of artefact peaks that do not represent the true mass of the parent molecule.
The use of cheap and miniaturised low resolution instruments for applications such as on-line and at-line monitoring also presents additional requirements; use in these environments inevitably results in the transition from use by skilled practitioners to users with lower skills and experience in mass spectrometry. As a result any methodology used to extract parent molecule masses from multiply charged mass-to-charge ratio spectra should be easy to use and require minimal user input.
A number of methods for extracting mass information from mass-to-charge ratio spectra with no isotopic resolution have been implemented. These are discussed below:
Zhang and Marshall describe a method ‘ZScore’ in the Journal of the American Society for Mass Spectrometry, vol. 9, 225-233 (1998). This method utilises peak picking and a scoring system based on the logarithm of the signal to threshold ratio. The ratio is calculated from background noise and a user defined signal to noise ratio. As this method is dependent upon peak picking, it is subject to the disadvantages of peak picking outlined previously. The need for the user to define a signal to noise ratio and a range of mass-to-charge ratios to calculate background noise is another disadvantage of this system. In particular, useful information may be lost if the noise level is set at an inappropriate level.
Morgner and Robinson describe the method ‘Massign’ in Analytical Chemistry, vol. 84, 2939-2948 (2012). Like the ZScore method, described above, this method is dependent upon accurate peak picking and user input in the form of two threshold levels. In addition, peaks with poor separation have to be identified manually for inclusion in the calculations. The method is therefore not well suited to extracting parent masses from low quality mass-to-charge ratio spectra.
In the International Journal of Mass Spectrometry, vol. 290, 1-8 (2010) and Analytical Chemistry, vol. 77, 111-119 (2005) Maleknia et al. describe a method ‘eCRAM’ which utilises the unique ratio of integers from charge states to calculate the charge states of peaks in a low resolution mass-to-charge ratio spectrum. This method is also dependent on clearly identifying peaks in the spectrum to be analysed.
Winkler describes a method ‘ESIprot’ in Rapid Communications in Mass Spectrometry, vol. 24, 285-294 (2010) which uses peaks observed in the mass-to-charge ratio spectrum to calculate the mass of the species at differing charge states. The correct charge states are identified by calculating the set of charge states which yield the lowest standard deviation with Bessel's correction. The performance of this method is also dependent upon the ability to clearly identify peaks of interest in the mass spectrum. The assumption that any peaks identified and used in the method are from a consecutive series of multiply charged ions is also a serious limitation of this method.
Mann et al. describe two methods in Analytical Chemistry, vol. 61, 1702-1708 (1989) which are also described in U.S. Pat. No. 5,130,538. The first method is an averaging algorithm which uses relative peak positions to infer charge states and calculate the mass of the parent molecule. This method is reliant on peak picking and has the potential to be strongly influenced by noise and extraneous peaks. A second method described in these two sources is a deconvolution algorithm. This method uses a transformation function to evaluate trial values of the parent molecule mass. This transformation function is calculated from the distribution function of the ion counts in the mass-to-charge ratio spectrum. This deconvolution method benefits from not being reliant on peak picking but, as they demonstrate in both references, is strongly influenced by background noise. This can result in the production of erroneous results, in the form of multiples or fractions of the parent molecule mass, appearing in the de-convoluted spectrum. An increase in background noise with mass in the de-convoluted spectrum is an additional undesirable artefact of this method.
A method utilising a deconvolution algorithm is also described in Analytical Chemistry, vol. 66, 1877-1883 (1994) and U.S. Pat. No. 5,352,891. Unlike the Mann algorithm described above ion counts are multiplied to together to form a multiplicative correlation algorithm rather than added together. This change in methodology reduces the impact of background noise and the introduction of artefact peaks. However, this multiplicative technique can be strongly disadvantaged by the presence of a single ion count equal to zero or an abnormally high ion count. The inclusion of this zero ion count to the calculation will lead to complete suppression of the signal in the de-convoluted spectrum. Similarly, the inclusion of a single large ion count (e.g. resulting from an adduct ion or noise) can unduly influence the signal in the de-convoluted spectrum.
The forward working maximum entropy method, first described by Reinhold et al. in the Journal of the American Society for Mass Spectrometry, vol. 3, 207-215 (1992), is advantageous as it has added discrimination against artefact peaks. However, the method can be computational expensive and extensive a priori knowledge of relative intensities and peak shapes in the mass-to-charge ratio spectrum is required for the effective extraction of the parent molecule mass.
What is required is a robust methodology which can extract the mass of multiply charged parent molecules from poor quality low resolution mass-to-charge ratio spectra, i.e. those without isotopic resolution, low signal to noise and containing extraneous adduct peaks. In addition, this methodology should not be reliant on the use of peak picking techniques, be strongly influenced by background noise and be prone to the generation of artefact peaks. The methodology should also require minimal user input and a priori knowledge; thereby allowing the successful implementation of the method by users who are not expert practitioners of mass spectrometry.